This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We investigated the mechanisms that lead to the production of pro-inflammatory mediators by human monocytes when these cells are exposed in vitro to live Borrelia burgdorferi (Bb) spirochetes. We first focused on myeloid differentiation primary response protein 88 (MyD88), an adapter molecule that is essential in the Toll-like receptor (TLR) pathway. Real-time PCR, flow cytometry and confocal microscopy experiments revealed that MyD88 was maximally expressed in THP-1 cells after 24-h stimulation of these cells with live Bb. Silencing of the MyD88 gene using small interfering RNA resulted in 24%, 35% and 84% down-modulation of the production of TNF-[unreadable], IL-8 and IL-6, respectively in THP-1 cells stimulated with live Bb. Specific silencing of the TLR1, TLR2 or TLR5 genes by RNA interference further revealed that silencing of the TLR1 and TLR2 genes alone or combined, but not the TLR5 gene caused a down-regulation of IL-6, IL-8 and TNF-[unreadable] in live Bb-stimulated THP-1 cells. Overall, similar results were obtained for THP-1 cells stimulated with purified lipoproteins. Our results indicate that the TLR pathway mediates at least in part the release of inflammatory mediators in human monocytes stimulated with live B. burgdorferi spirochetes, and further suggest that the TLR-dependent interaction between these cells and live spirochetes is mediated by spirochetal lipoproteins but not flagellin.